Comparison of patterns of hybrid resistance to the BALB/c plasmacytomas LPC-1 and MPC-11

Summary Patterns of genetic control of hybrid resistance to the BALB/c plasmacytoma LPC-1 were studied for comparison with those to MPC-11, a plasmacytoma investigated previously. The overall patterns of hybrid resistance to the two tumors were similar, i.e., hybrids between BALB/c and BALB congenic resistant (CR) strains, A and A CR strains, SJL and DBA/2 were as susceptible to LPC-1 as BALB/c mice themselves, whereas hybrids between BALB/c and AKR, C57BL/Ks, DBA/1, C57BL/6 (B6), C57BL/10 (B10) and B10 CR strains were resistant to LPC-1 as previously shown with MPC-11. Heterozygosity within the H-2 complex alone was insufficient for resistance to either tumor. Among hybrids between BALB/c and the B10 CR strains, however, the presence of certain H-2 haplotypes influenced the degree of resistance seen and this H-2 effect was different for the two tumors. A sex effect on resistance to LPC-1, but not to MPC-11, was seen among F₁ hybrids between BALB/c and DBA/1 although not in any other F₁ hybrids. Among ((B10×BALB/c)F₁×BALB/c) and (BALB/c×(B10×BALB/c)F₁) and ((BALB/c×B10)F₁×BALB/c) and ((BALB/c×B10)F₁×BALB/c) backcross mice, however, significantly more males than females were resistant to LPC-1 and the results of this study are compatible with the idea that in F₁ hybrids between BALB/c and B10, resistance to LPC-1 is controlled by two dominant autosomal genes, one of which is sex-limited and neither of which is linked to H-2. In contrast, hybrid resistance to MPC-11 in this cross is controlled by a single gene. Cross-protection experiments indicated that the two tumors share at least one tumor-associated transplantation antigen.     

Immune response to the H-X antigen on P815-X2

In a preceding report, the detection of an H-2-linked immune response to the H-X ^d antigen on the P815-X2 mastocytoma was demonstrated by the significantly increased survival of (C57BL/6 × DBA/2)F₁ (B6D2F1) male hybrids (H-X ^b ) compared with female siblings (H-X ^{b/H-X d} ) after injection with the histocompatible tumor (H-X ^d ). This interpretation was supported by the absence of this sex effect in reciprocal D2B6F1 hybrids (H-X ^d and H-X ^{d/H-X b} ). Additional findings presented in this paper support the conclusion that this sex effect is due to a true immunological response to H-X ^d : (a) Reciprocal (DBA/2 × C57BL/6 H-2 mutant)F₁ hybrids, as well as D2B6F1, failed to exhibit the sex effect: (b) the demonstration of the sex effect in (BALB/c × DBA/2)F₁ and (BALB/c-H-2 ^dm2 × DBA/2)F₁ hybrids and in (C57BL/10 × DBA/2)F₁ hybrids was consistent with the known H-X incompatibilities between the strains BALB/c and DBA/2 and C57BL/10 and DBA/2, respectively, previously demonstrated by skin grafting; and (c) the sex effect was not abrogated by castration of male B6D2F1 hybrids. Variability in the presence or absence of the sex effect was observed in various [recombinant inbred (RI) × DBA/2]F₁ hybrids and may be attributed to the influence of a regulatory non-H-2 gene which is closely linked to the gene coding for mouse kidney-androgen-regulated protein (KAP) but androgen-independent, or to variability in inheritance of the H-X ^b allele among the RI lines. It is proposed that the P815-X2 model may be utilized to type RI lines derived from a cross between C57BL/6 and DBA/2 for their H-X genotypes.Abbreviations B C57BL/6 origin allele - B6 C57BL/6 - B10 C57BL/10 - B6D2F1 (C57BL/6 × DBA/2)F₁ - B6 ^m D2F1 (C57BL/6 H-2 mutant × DBA/2)F₁ - bm10 B6.C-H-2 ^bm10 - C BALB/c - D DBA/2 origin allele - D2 DBA/2 - dm2 BALB/c-H-2 ^dm2 - H-X X chromosome-determined histocompatibility antigen of the mouse - Ir gene, immune response gene - KAP kidney androgenregulated protein - MST median survival time - RI recombinant inbred - SDP strain distribution pattern     

Genetic and functional analyses of the primary in vitro CTL: Response of NZB lymphocytes toH-2-compatible cells

Spleen cells from NZB mice make an unexpected primary cytotoxic T lymphocyte (CTL) response to BALB/c cells in vitro. In this study, it is shown that this response is comprised of at least three independent components. These include a response to antigens recognized in association with H-2^d products, a response to Qa-1^b-associated antigens which is notH-2-restricted and a response directed toward antigens not associated with either H-2^d- or Qa-1^b-coded determinants. The last response appears to be the weakest of the three. In addition, cells from NZB F₁ mice which were either homozygous (Qa-1 ^a /Qa-1 ^a ) or heterozygous (Qa-1 ^a /Qa-1 ^b ) forQa-1 alleles, all responded to BALB/c cells. These data suggest that the NZB CTL response to BALB/c cells is not solely dependent on antigens coded for by genes in theH-2D-Tla region for either the sensitization or effector phases of the response. The ontogeny of the NZB anti-BALB/c CTL response coincides with that of a number of B-cell abnormalities but is shown in experiments with-suppressed NZB mice to be independent of B-cell dysfunction. Studies with (NZB x B10.D2)F₁ + B10.D2 mice demonstrated that the anti-BALB/cCTL response to antigens coded for outside ofQa-1 is governed by at least two genes. Finally, it is shown that another conventionallyH-2-restricted response, that to TNP-modified isologous cells, is neither significantly cross-reactive nor markedly elevated in NZB mice. — The foregoing observations suggest that some subsets of NZB T lymphocytes are intrinsically abnormal. The possibilities that the apparent hyperreactivity of NZB CTL precursors, evidenced in the response to BALB/c cells, is primary or results from the secondary effects of excess T-cell help are discussed.     

The regulation of immune responses to multiple non-H-2 antigens on tumor cells: I. Differential responses of BALB/c F1 hybrids to the P815 mastocytoma in vivo

Factors influencing host resistance to the growth of a tumor bearing many mismatched minor histocompatibility antigens (MiHA) were studied. BALB/c (H-2^d) and several of its F₁ hybrids were injected intraperitoneally with DBA/2 (H-2^d) P815 tumor cells. Compared to BALB/c, which was moderately susceptible, F₁ hybrids of BALB/c with CBA, AKR, C3H.OH, and BIO H-2-congenic strains were highly susceptible, whereas hybrids of BALB/ c with A, A.SW, and BALB.B strains were quite resistant. Susceptibility was observed only with the intraperitoneally injected tumor, since both BALB/c and (CBA x BALB/c)F₁ were resistant to the same tumor injected subcutaneously, and survival times of DBA/2 skin grafts did not differ between susceptible and resistant strains. Susceptibility was in part a function of the number of MiHA incompatibilities between tumor and host although the specific loci involved could not be identified. For example, susceptible (CBA x BALB/c)F₁ hybrids probably shared certain MiHA with DBA/2 which BALB/c lacked, and which therefore subtracted from the net antigenic strength of the tumor in the hybrid, compared to its strength in BALB/ c. This interpretation was supported by in vitro studies which confirmed that the susceptible hybrids shared more MiHA with DBA/2, than did the resistant hybrids. Resistance was at least partially regulated by the host H-2 genotype, as shown by the observation that (BALB/ c x BALB.B)F₁ (H-2^d/b) mice were significantly more resistant than BALB/c. Segregation studies of the resistant (BALB/c x A)F₁ hybrids, indicated that in addition to H-2, a nonH-2 gene in the A background was operating to confer resistance. Thus the factors influencing susceptibility to the MiHA-incompatible tumor were: (i) site of injection; (ii) the combined strength of the disparate MiHA; (iii) the host H-2 genotype; and (iv) at least one host nonH-2 gene conferring increased responsiveness.     

Transfer of long-lasting tumor immunity by immune T cells from MHC congenic mice: Migration,survival and tumor-protectivity of cytotoxic donor cells

Immunocompetent B10.D2 (H-2^d) mice are able to reject the highly malignant lymphoma ESb of DBA/2 (H-2^d) origin very effectively. Seven days after intravenous injection of the ESb tumor cells, B10.D2 mice developed a strong tumor-rejection response which was associated with the generation of anti-tumor T cells in their spleens with direct cytotoxic activity. Most of the cytotoxic potential was directed against the minor histocompatibility differences as demonstrated by the lysis of unrelated DBA/2 derived Eb tumor cells and normal DBA/2 but no B10.D2 derived ConA lymphoblasts. A previously performed clonal analysis, however, revealed a minority population of CTL clones which specifically recognized the ESb specific transplantation antigen (ESb-TATA). When transferred systemically into DBA/2 mice, the B10.D2 anti-ESb immune spleen cells could delay the outgrowth of s.c. transplanted ESb tumor cells. When the ESb tumor cells were experimentally distributed in a s.c. implanted sponge-matrix, the i.v. injected B10.D2 immune cells could confer complete protective immunity against the metastatic tumor, provided the recipients were pre-treated with 5 Gy to allow a better take of the allogeneic cells. The distribution of intravenously injected B 10D2 donor spleen cells was assessed in the recipients up to 50 days by cytotoxicity testing and assaying for the expression of the ₂ microglobulin allelic form b ( ₂m^b). These tests revealed a high propensity of donor cells to populate the spleen and lymph nodes of the DBA/2 recipients. Again this was particularly marked in sublethally irradiated mice where a long-lasting lymphoid chimerism was established.     

Increased immunogenicity of L1210 leukemia following short-term exposure to 5(3,3′-dimethyl-1-triazeno)-imidazole-4-carboxamide (DTIC) in vivo or in vitro

Summary Short-term exposure of L1210 Ha leukemia to DTIC in vivo or in vitro resulted in the generation of leukemic cells that were moderately immunogenic for histocompatible (BALB/C × DBA/2)F₁ (CD2F₁) mice. In vivo treatment was carried out in the peritoneal cavity of CD2F₁ host for 8–36 h. In vitro experiments were performed in glass vessels, in which tumor cells were incubated with DTIC for 2 h at 37° C. The in vitro generation of immunogenic leukemia was conditioned by the presence of mouse liver microsomes capable of producing metabolic transformation of DTIC. It follows that the increase of tumor cell immunogenicity produced in vitro and possibly in vivo by DTIC is due to (a) metabolic product (s) that has (have) not yet been identified. Somatic mutation, selection, viral activation, or other mechanisms could be responsible for the DTIC effect. The present studies suggest that similar in vivo or in vitro techniques could be used to obtain human tumor cells with higher immunogenicity.Abbreviations AIC 5-aminoimidazole-4-carboxamide - BCNU 1,3-bis-chloroethyl nitrosourea - Cy of cyclophosphamide - DMITI drug-mediated increase tumor immunogenicity - DTIC 5(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide - MLP mouse liver preparation - PB phenobarbital     

Experimental allergic orchitis in mice

Inbred strains of mice were studied for their susceptibility to the induction of experimental allergic orchitis after sensitization with mouse testicular homogenate in complete Freund's adjuvant accompanied by injections of extract from Bordetella pertussis. Susceptibility to autoimmune orchitis was found to be linked to the major histocompatibility complex in BALB/c and C57BL/10 mice and mapped to genes encoded within the H-2D ^dregion. In five of six groups of bidirectional (susceptible × resistant) F₁ hybrids, H-2D ^d-linked susceptibility was inherited as a dominant autosomal trait. However, in (BALB/cByJ × DBA/2J)F₁ and (DBA/2J × BALB/cByJ)F₁ hybrids, dominant autosomal resistance to the induction of autoimmune orchitis was observed. Backcross analysis between the resistant F₁ hybrid and the susceptible BALB/cByJ parent suggests that a single independently segregating DBA/2J locus is capable of negating H-2D ^d-linked susceptibility, and controls resistance to the induction of autoimmune orchitis.Abbreviations used in this paper BP extract Bordetella pertussis extract - CFA complete Freund's adjuvant - EAO experimental allergic orchitis - Ir immune response - MHC major histocompatibility complex - MLH mouse liver homogenate - MTH mouse testis homogenate - PI pathology index     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

Teratocarcinoma transplantation rejection loci: AnH-2-linked tumor rejection locus

Embryoid bodies (ascites tumor) from a 129/Sv transplantable teratocarcinoma produce tumors (100%) in syngenic 129/Sv mice but fail to form tumors (3–6%) in BALB/c mice, C3H/He mice and C57BL/6 mice, in spite of the fact that the malignant stem cells of this tumor do not express detectable H-2 antigens. The available evidence indicates that this allogeneic tumor restriction has an immunological basis; 100% of the F₁ hybrid mice between 129/Sv and the three other inbred mouse strains accept the 129/Sv teratocarcinoma. The backcross and F₂ mice segregate the BALB/c, C3H/He and C57BL/6 tumor transplantation rejection loci in a manner that indicates that each of these inbred strains of mice contain one to two major transplantation rejection loci. A linkage analysis in the BALB/c and C3H/He backcross and F₂ generations indicates that these mice have a teratocarcinoma transplantation rejection locus on chromosome 17, about eight to nine recombination units from theH- 2 complex. An F₁ complementation analysis between allogeneic mice that each reject teratocarcinomas tumors (BALB/c × C57BL/6 and C3H/He × C57BL/6), indicates that the C57BL/6 mice have the 129/Sv tumor-accepting (sensitive) allele at theH-2-linked locus but reject teratocarcinomas because of antigenic differences at a second locus.While these major teratocarcinoma transplantation rejection loci determine the acceptance or rejection of a tumor by a mouse injected with high doses of tumor tissue (750 g of tumor protein), evidence is presented for a number of minor genetic factors that can (1) affect the efficiency of tumor rejection and (2) cause complete tumor rejection at lower tumor doses (7.5–75 g of tumor protein).     

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.     

Production of specific macrophage-arming factor precedes cytotoxic T lymphocyte activity in vivo during tumor rejection

Summary Recently we published a hypothesis on the immunological events occurring during tumor rejection. One of the implications of this hypothesis is that specific macrophage-arming factor (SMAF) is produced early during the initiation of the immune response, whereas the classical cell-mediated immune response components, such as cytotoxic T lymphocytes (CTL), are produced later, that is, during the amplifier-effector phase. In this paper we establish the kinetics of the induction of (a) lymphocytes producing SMAF and (b) CTL. Groups of DBA/2 mice were injected i.p. once, twice or three times with irradiated and/or non-irradiated syngeneic SL2 tumor cells, the injections being given at intervals of 10 days. After each of these injections the production of SMAF and the expression of CTL activity were established. The results showed that in the peritoneal cavity SMAF-producing lymphocytes appeared earlier than cytotoxic lymphocytes (CTL). In addition, it was shown (a) that SMAF does not interfere with the in vitro cytotoxicity expressed by CTL and (b) that in addition to CTL memory cells, SMAF-producing memory cells were also induced after injection of syngeneic tumor cells. These data support the hypothesis that SMAF is involved in the early phase of the cellular immune response against tumors, whereas CTL are induced later.     

Interacting genetic factors controlling the antibody response against the H-2.2 specificity

The antibody response against the H-2.2 specificity has been studied in three H-2 ^d strains, B10.D2, DBA/2, and BALB/c, and their hybrids (B10.D2 × DBA/2)F₁ and (B10.D2 × BALB/c)F₁. The genetic control of the response appears to be complex: The three pure strains are responders, whereas both hybrids when immunized with C3H-HTG are nonresponders. Individual analysis of N3 offspring is compatible with the idea that, in this combination, an Ea-4 incompatibility between donor and immunized strain is necessary for the anti-H-2.2 response to occur. H-2 ^d /H-2 ^k hybrids (B10.BR × B10.D2)F₁ or (B10.BR × DBA/2)F₁ are responders when immunized with C57BL/10 (H-2 ^b ) but not with B10.A(2R) (H-2 ^h ), indicating that simultaneously recognized H-2 specificities are necessary for the anti-H-2.2 response.     

No recombinations between Tcra-V and Tcra-C gene segments in 669 backcross mice

Because T-cell receptor (Tcr) genes may possibly function as non-major histocompatibility complex (MHC) immune response genes or predispose for autoimmune diseases, it is important to know how these genes are inherited. We found that Bgl I-digested DNA of BALB/c, C3H, DBA/2, and C57BL/6 exhibited restriction enzyme fragment length polymorphisms (RFLPs) for the Tcra-V1, Tcra-V2, Tcra-V4, Tcra-V6, Tcra-V7, Tcra-V8, Tcra-V11, Tcra-122, Tcra-V13, and Tcra-C gene segments. Inheritance of these RFLPs in 669 offspring from (BALB/c × C57BL/6) × BALB/c, (BALB/c × C57BL/6) × C57BL/6, (C57BL/6 × DBA2) × DBA/2, and (C57BL/6 × C3H) × C3H backcrosses was studied. Since we did not find any recombinations in the offspring, Tcra-V and Tcra-C gene segments are tightly linked and inherited as a haplotype. A peculiar finding was that 22 out of 103 (BALB/c × C57BL/6) × BALB/c offspring, heterozygous for Tcra-C, had deleted a C57BL/6 Tcra-V1 band as well as Tcra-V2 and Tcra-V4 bands. As will be discussed, this deletion is probably caused by heterogeneity in the C57BL/6 breeding stock of a commercial supplier. In seven BXD and BXH recombinant inbred strains with known recombinations between the Tcra-C and Es-10 loci, all Tcra-V RFLPs cosegregated with the Tcra-C RFLP. This finding agrees with the conclusion from our backcross studies; namely that Tcra-V and Tcra-C gene segments are tightly linked.     

Alloantigen-induced cytotoxicity against syngeneic tumor cells: analysis at the clonal level

It has been shown that peritoneal exudate cells (PEC) from BALB/c mice immunized with minor histocompatibility antigens presented by DBA/2 or B10.D2 spleen cells are capable of lysing syngeneic YC8 tumor cells in a 4-hr 51Cr-release assay. In this study, we employed limiting dilution analysis to determine the frequency of CTL precursors (CTL-P) reactive against both the specific DBA/2 (or P815) target and the syngeneic tumor YC8. The mean frequency of anti-DBA/2 CTL-P in PEC from BALB/c mice immunized with DBA/2 was 1/302. Between one-third and one-fifth of limiting dilution microcultures that exhibited lytic activity against DBA/2 lymphoblasts (or P815) were also able to lyse YC8. No lysis of YC8 was observed in the absence of a parallel lysis on DBA/2 lymphoblasts or P815 target cells. T cell clones, derived by micromanipulation from microcultures selected for cytotoxic activity against YC8 and/or P815, maintained either the specific anti-allogeneic or the doubly reactive ( antiallogeneic plus anti-syngeneic tumor) phenotype. Fourteen clones (six specific and eight doubly reactive) were tested for cytotoxic activity on a panel of target cells with different haplotypes. All showed H-2-restricted specificity for minor histocompatibility antigens shared by DBA/2 and B10.D2. The restriction element for some of the clones mapped in the K region of the H-2 complex, whereas for other clones the restriction element mapped in the D region; both K- and D-restricted clones were able to lyse YC8. When the clones that exhibited lysis on YC8 were tested on two other BALB/c tumor targets, LSTRA, a Moloney virus induced lymphoma, and RL male-1, a radiation induced lymphoma, two of seven were found to lyse all three syngeneic tumor targets equally well, but not syngeneic BALB/c blasts. These clones were functionally categorized as conventional CTL because they were unable to proliferate when cultured with antigen in the absence of exogenous lymphokines, and were unable to produce lymphokine with IL 2 activity when stimulated by the appropriate splenocytes. When tested in vivo in a Winn assay, a strong anti-tumor activity against YC8 was exerted by the anti-DBA/2 clones DY4 -3 and DY16 -3. These clones lysed both YC8 and the immunizing target cells in vitro. No in vivo effect in neutralizing YC8 tumor growth was observed with clone D2-1, a clone that lysed DBA/2 targets but not YC8 in vitro.     

Consequences of a single Ir-gene defect for the pathogenesis of lymphocytic choriomeningitis

The H-2L ^d allele has been identified by others as the sole Ir gene in the H-2 ^d haplotype for the cytotoxic T lymphocyte (CTL) response to mouse lymphocytic choriomeningitis virus (LCMV). The BALB/c-H-2 ^dm2 (C-H-2 ^dm2 ) mutant lacks H-2L ^d , and thus should be ideal for assessing the contribution of virus-immune CTL to LCM immunopathology. Comparison of the C-H-2 dm2 mice with congenic BALB/c mice revealed that there is a delay of about 24 h in the onset of severe inflammatory process and symptoms in the mutant strain, but the absence of H-2L ^d did not prevent the later development of fatal disease in mice injected intracerebrally (i.e.) with neurotropic LCMV. This could indicate that virus-immune CTL are not the major mediators of clinical LCM. Spleen cells from LCMV-primed BALB/c mice did not show CTL activity for LCMV-infected C3H.OH, C-H-2 ^dm2 , or (CBA × C-H-2 ^dm2 )F₁ target cells. However, immune lymphocytes from both the mutant and the F₁ strains lyse virus-infected BALB/c cells. Furthermore, BtO.HTG and, in some experiments, B10.A(5R) mice generated CTL lytic for LCMV-infected BALB/c, C-H-2 ^dm2 , and (CBA × CH-2 ^dm2 )F₁ macrophages. Apparently H-2L ^d is immunodominant in the H-2^d restricted response to LCMV. However, in the absence of H-2L ^d , it seems that H-2K ^d and, to a lesser extent, H-2D ^d also serve as Ir genes for the CTL response in this infection. Even so, the absence of the H-2L^d-restricting element results in a disease process which is either delayed in onset or less severe.     

Purification and characterization of the Danish (skive) variant of mouse liver alcohol dehydrogenase

The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the variant and normal enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The K _m and V _max of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.This work was supported by NIAAA Grant AA-04307.     

Evaluation of in vivo and in vitro effectivity of immune defense against a spontaneously arising,nonlymphoid rat tumor

Summary It was evaluated in vitro whether it is possible to induce immunogenicity by haptenization of a nonlymphoid rat tumor, BSp6AS, which is known to be antigenic, but non-immunogenic, in the syngeneic host. The effectivity of induced immunogenicity was tested in vivo.BSp6AS, an NK- and macrophage-resistant variant of a spontaneously arising fibrosarcoma in the BDX rat strain, does not induce a primary or a secondary T cell response after in vivo or in vitro priming. This deficiency in cytotoxic response is due solely to failure of activation of helper T cells (T_H), since (a) cytotoxic T cells (CTL) can be detected after in vitro stimulation in the presence of interleukin 2 (IL-2)-containing medium; and (b) there are no indications for down-regulation of a potential specific immune response by suppressor T cells (T_S). The lack of activation of tumor-specific T_H can be bypassed by activation of hapten-specific T_H. Upon coculture with haptenized tumor cells as a stimulator population, both hapten-specific and tumor-specific CTL are activated by hapten-specific T_H.In line with the findings in vitro, no transplant rejection of naive tumor cells was seen after a variety of immunization schedules. But immunized F₁ hybrids did reject tumor grafts, supporting the hypothesis of lacking help in the syngeneic situation. This could be confirmed in the syngeneic system by adoptive transfer experiments. Tumor-specific CTL, educated in vitro in the presence of IL-2, were ineffective. But complete protection against haptenized, and partial protection against native tumor cells was achieved in the additional presence of hapten-specific T_H.To our knowledge these experiments prove for the first time that hapten-specific T_H are efficient in inducing an immune response even against a nonlymphoid, nonimmunogenic tumor, i.e., it is possible (a) to activate tumor-specific CTL and (b) to initiate tumor graft rejection via hapten-specific T_H.Abbreviations BSS Hank's balanced salt solution - CFA complete Freund's adjuvant - ConA Concanavalin A - CTL cytotoxic T cell - iFA incomplete FA - IL-2 interleukin 2-containing supernatant of rat spleen cells cultured in ConA - IP intraperitoneal - IV intravenous - LNC lymph node cells - 2-ME 2-mercaptoethanol - MHC major histocompatibility complex - MLTC mixed lymphocyte tumor cell culture - RPMI 1640 supplemented with antibiotics, L-glutamine and fetal calf serum - SC spleen cells - SC subcutaneous - TAA tumor-associated antigen(s) - TB tumor bearer - TD₁₀₀ tumor cell dose resulting in 100% takes - T_H helper T cells - T_S suppressor T cells - TNBS trinitrobenzenesulfonic acid - TNP trinitrophenyl - WAB Wistar Nottingham rats     

Measles Virus DNA Vaccination: Antibody Isotype Is Determined by the Method of Immunization and by the Nature of both the Antigen and the Coimmunized Antigen

Plasmids encoding the measles virus hemagglutinin (HA) and nucleoprotein (NP) proteins inoculated into the skin of BALB/c mice by the gene gun method induced both humoral and cytotoxic lymphocyte class I-restrict- ed immune responses. Although intramuscular immunization induces the immunoglobulin G2a (IgG2a) antibody isotype for both antigens, with gene gun immunization, the NP still generated mainly IgG2a and the major isotype induced by the HA was IgG1. Interestingly, gene gun coimmunization of HA and NP plasmids resulted in a dominant IgG1 HA response and the switching of antibodies generated against the NP to the IgG1 isotype.The initial studies showing that injection of DNA into muscle induces an immune response to the encoded protein opened a new approach to vaccination (for reviews, see references 19 and 22). Recent studies suggest that inoculated muscle cells probably act only as a source of antigen and that immune priming takes place elsewhere in the body (14). For example, excision of an injected muscle a few minutes after DNA inoculation did not affect antibody and cytotoxic T-lymphocyte (CTL) responses (21). Thus, it may be interesting to examine other DNA delivery systems to study how the immune system responds to DNA vaccination. One alternative system involves precipitating DNA onto gold beads which are then propelled into the skin by means of pressurized helium gas (12). When such a system is used, less DNA is required, but unlike the case with intramuscular inoculations, the response is Th2-like, generating immunoglobulin G1 (IgG1) antibodies (17). More recent observations suggest that this is probably due to the mode of inoculation rather than the route (10). We have been studying DNA vaccination against the paramyxovirus measles virus (MV). This disease is one of the primary causes of infant mortality in developing countries, and there is an urgent need for an effective vaccine in infants, as the present live attenuated vaccine is inefficient in the presence of maternal antibodies. Our previous studies established that in a mouse model at least three MV proteins play a role in protection (23). Both glycoproteins, hemagglutinin (HA) and fusion, induce neutralizing antibodies (9, 11), and HA and nucleoprotein (NP) induce CTLs (3, 4), which do not protect against infection but help in recovery (5). In our previous study on DNA vaccination, we showed that intramuscular inoculation of DNAs coding for the MV HA and NP (pV1J-HA and pV1J-NP [6]) induced class I-restricted CTLs and a humoral response corresponding to a Th1 response (6). In the present study, we have extended our observations to compare the same plasmids’ ability to induce an immune response when they are delivered into the skin by a gene gun (Bio-Rad, Ivry sur Seine, France). Gold beads were coated with DNA as follows: approximately 30 mg of gold powder (1.0-μm gold beads; Bio-Rad) was mixed with 100 μl of 0.1 M spermidine (Sigma, L’Isle D’Abeau, France). After sonication, 0.5, 2, or 5 μg of plasmid DNA was added per mg of gold powder, and then 200 μl of 2.5 M CaCl₂ was added to the mixture, with gentle vortexing. Pellets were washed three times and suspended in cold 100% ethanol. Tubes containing dried DNA-coated gold beads were stored at 4°C.

Immune response to MV HA DNA.

Six- to eight-week-old female BALB/c mice (Iffa-Credo, Domaine des Oncins, France) were immunized via the shaved abdominal epidermis one to three times at 21-day intervals with 0.5, 2, or 5 μg of pV1J-HA DNA/mg of gold beads. Two gene gun inoculations (each containing 0.5 mg of gold beads) were given for each dose. The antibody levels measured by enzyme-linked immunosorbent assay, as previously described (6), reached a plateau after two inoculations and did not significantly increase with a third inoculation (result not shown).Our previous studies with intramuscular inoculation established that pV1J-HA induced IgG2a antibodies which are associated with a Th1-type response. When we studied the antibody isotype induced in BALB/c by the gene gun immunization, we observed that it was mainly IgG1 (Fig. ​(Fig.1).1). These data are similar to those described for influenza hemagglutinin by Feltquate et al. (10). The antibody isotype did not vary with time after immunization, number of immunizations, or the amount of plasmid used (data not shown) and was not influenced by genetic background, as pV1J-HA-immunized DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice induced mainly the IgG1 isotype (Fig. ​(Fig.11).Open in a separate windowFIG. 1Anti-MV HA isotype of antibodies induced in BALB/c, DBA/2 (H-2^d), C3H (H-2^k), and C57/Black (H-2^b) mice immunized with 0.5, 2, or 5 μg of pV1J-HA by epidermal gene gun. Sera were collected 3 weeks after the immunization. Sera from mice immunized with a control pV1J had means ± standard deviations of 158 ± 198 ng/ml for IgG1 anti-HA antibodies (n = 11) and 10 ± 18 ng/ml for IgG2a anti-HA antibodies (n = 11). Data represent individual animals. To study CTL activity, spleen cells from the immunized mice were stimulated in vitro and analyzed in a cytolytic assay as previously described (6). Despite the apparent Th2-type response, good memory CTL responses were obtained with all protocols used, even when responses were measured just 8 days after a single immunization (Fig. ​(Fig.2),2), and persisted for several months. Open in a separate windowFIG. 2Anti-MV HA and NP CTL response after immunization with pV1J-HA or -NP, respectively. BALB/c mice were immunized with 0.5 (circle), 2 (triangle), or 5 (square) μg of pV1J-HA by epidermal gene gun one (a, d), two (b, e), or three (c, f) times at 3-week intervals. The spleen cells were removed 3 weeks (continuous line) or 8 days (dotted line) after the last immunization. After in vitro stimulation with P815-HA or -NP cells, respectively, lysis was measured on P815-HA or -NP cells, and P815 cells were used as a negative control. The results show the specific lysis of targets at graded effector/target ratios. Each curve represents an individual animal.

Immune response to MV NP DNA.

BALB/c mice were immunized with pV1J-NP with the gene gun and a similar schedule of immunizations. The antibody response with the different number of doses and different plasmid concentrations was similar to that observed for HA, i.e., increased levels after one boost. Similar antibody levels were induced in the range of 0.5 to 5 μg of DNA (data not shown). As was previously shown by intramuscular inoculation (6), the antibody isotype induced was mainly IgG2a (Fig. ​(Fig.3),3), in contrast to the HA results. One explanation for this could be that as the NP is a cytosolic protein and the HA is membrane bound, the potential processing and presentation of the two proteins may be different. However, the same argument would be valid for intramuscular inoculation. Furthermore, it has been reported that gene gun immunization with influenza NP induces a Th2 response (17), so clearly the directed differentiation of T cells is more complicated than a simple distinction between cytosol and membrane-bound proteins. The two methods of immunization (intramuscular versus gene gun) target different cell types, possibly influencing the T-cell response. Furthermore, 9 weeks after immunization, one-third of the 18 mice analyzed showed increased levels of anti-NP IgG1 over IgG2a, regardless of the quantity of DNA injected or the number of inoculations (data not shown). CTL responses were also high, even after a single inoculation (Fig. ​(Fig.2).2). Open in a separate windowFIG. 3Anti-MV NP antibody response as measured by enzyme-linked immunosorbent assay in BALB/c mice immunized with 5 μg of pV1J-NP by epidermal gene gun. Sera were collected 3 weeks after immunization. Each pair of bars represents an individual animal. Sera from mice immunized with a control pV1J had means ± standard deviations of 13 ± 45 ng/ml of IgG1 anti-NP antibodies (n = 11) and 83 ± 276 ng/ml of IgG2a anti-NP antibodies (n = 11).

Coimmunization of HA and NP DNA.

Our results show that when injected by the gene gun, the different MV proteins induce different antibody isotypes. This phenomenon has been suggested to parallel the induction of Th1 and Th2 pathways (1). The pathway taken has been shown to be influenced by the induction of certain cytokines. To determine if coimmunization of these two plasmids would influence the isotype of the antibody response, BALB/c mice were immunized with a mixture of pV1J-HA and pV1J-NP in ratios of 1:1, 4:1, or 1:4 while the total amount of DNA was kept constant (5 μg).Measurement of the anti-HA isotype antibody in mice vaccinated with the different mixtures showed it to be mainly IgG1, similar to that for HA alone (data not shown). In contrast, the anti-NP antibodies switched from the IgG2a to the IgG1 isotype after coimmunization (Fig. ​(Fig.4).4). The proximity of expression of the two antigens was not important in this switching effect, as when pV1J-HA and -NP were inoculated separately in different areas of the skin, the antibody response induced 3 weeks later was the same as that induced when the mixture was inoculated (Fig. ​(Fig.4).4). When analyzed 6 weeks later, only one of six mice showed IgG2a predominance. Open in a separate windowFIG. 4Relationship between the isotype of anti-NP antibodies in sera from mice immunized with 5 μg of pV1J-NP or mixtures of pV1J-HA and pV1J-NP at ratios of 1:1, 4:1, and 1:4 so that the total quantity of DNA/mg gold beads was 5 μg, or pV1J-HA and pV1J-NP injected in different skin area. BALB/c mice were immunized by epidermal gene gun. Sera were collected 3 weeks after immunization. Data are results for individual animals.Cytokines have been used to direct the immune response in several studies. Expression of interleukin-12 either alone or with immunizing antigens can increase protection against microbial pathogens (2) or tumors in animal models (13, 18), in parallel with a Th1 response. Expression or addition of interleukin-4 with the immunogen induces a Th2 response (16, 20). The local concentrations of the cytokines in the initial priming of the immune response are probably critical, as once the T cells have been committed, they cannot be modified. Although some studies have suggested the possibility of Th1 and Th2 switching, a more recent study has shown that once differentiated, T cells cannot switch (15). In agreement with this, Feltquate et al. (10) have shown that initial immunization establishes the Th-cell type of the immune response and that this is not modified by subsequent alternative methods of immunization.Acute viral infections induce a Th1 response, whereas soluble proteins favor a Th2 response (7). When tetanus toxoid was administered 1 day after viral infection, the response to this soluble protein changed from Th2 to Th1 (8). Presumably, this change is due to the domination by the cytokines induced by the viral infection of those produced by the tetanus toxoid. In our studies, we observed that after the coexpression of MV HA and NP, the HA-induced Th2 response was dominant. These observations will obviously have an impact on DNA vaccination, as DNAs coding for several pathogens should ideally be administered concomitantly.     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.